牛Bta-miR-494序列特征及其组织表达分析

doi:10.7668/hbnxb.20194709

摘要/Abstract

摘要：

为探究牛Bta-miR-494的序列特征及其组织表达情况,运用生物信息学方法分析其上游序列的 CpG岛、启动子及转录因子特征及其在不同物种间的保守性;之后对牛Bta-miR-494的靶基因进行预测及功能富集分析;最后通过qRT-PCR方法检测其在牛不同组织及不同时期同一组织间的时空表达特征。结果表明,Bta-miR-494在不同物种间高度保守且与山羊亲缘关系最近,其上游不含CpG岛、存在2个转录起始位点;预测靶基因富集分析表明,Bta-miR-494的靶基因显著富集在PI3K-Akt、p53及MAPK等与肌肉生长发育及代谢相关的信号通路;qRT-PCR结果显示,Bta-miR-494在成年牛肝脏中的表达量最高,睾丸中表达量最低;且Bta-miR-494在成年牛股二头肌中的表达量显著高于初生犊牛,而心肌和背最长肌中的表达量显著低于初生犊牛。这种组织间差异性表达提示,Bta-miR-494可能在牛不同生长阶段的肌肉组织发育过程中主导不同的生肌表达模式。

关键词: 牛, Bta-miR-494, 功能富集, 组织表达, 骨骼肌

Abstract:

In order to investigate the sequence characteristics of bovine Bta-miR-494 and its tissue expression,bioinformatics methods were used to analyze the characteristics of CpG islands,promoters and transcription factors of its upstream sequence and their conservatism among different species;then predicted and functionally enriched the target genes of bovine Bta-miR-494;and finally detected its spatial and temporal expression characteristics among different tissues and the same tissue at different periods of time of the bovine Bta-miR-494 by qRT-PCR method.The results showed that Bta-miR-494 was highly conserved among different species and had the closest affinity to goat,with no CpG island upstream and two transcriptional start sites;the target gene enrichment analysis showed that the target genes of Bta-miR-494 were significantly enriched in the signaling pathways related to muscle growth and metabolism,such as PI3K-Akt,p53 and MAPK; the predictive target gene enrichment analysis showed that the target genes of Bta-miR-494 were significantly enriched in the signaling pathways related to muscle growth and metabolism.qRT-PCR results showed that the expression of Bta-miR-494 was highest in the liver and lowest in the testis of adult cows;and the expression of Bta-miR-494 was significantly higher in the biceps femoris of adult cows than that of newborn calves,whereas the expression of Bta-miR-494 in the cardiac muscle and longitudinal muscle of dorsal cows was significantly lower than that of newborn calves,while the expression of Bta-miR-494 in the cardiac muscle and the longissimus dorsi muscle was significantly lower than that of newborn calves.This inter-tissue differential expression suggests that Bta-miR-494 may dominate different myogenic expression patterns during the development of muscle tissues at different growth stages in cattle.

Key words: Bovine, Bta-miR-494, Functional enrichment, Tissue expression, Skeletal muscle

徐俊杰, 丁燕玲, 李承隆, 周小南, 苏总华, 曲畅, 史远刚, 康晓龙. 牛Bta-miR-494序列特征及其组织表达分析[J]. 华北农学报, 2024, 39(3): 215-223. doi: 10.7668/hbnxb.20194709.

XU Junjie, DING Yanling, LI Chenglong, ZHOU Xiaonan, SU Zonghua, QU Chang, SHI Yuangang, KANG Xiaolong. Sequence Characterization and Tissue Expression Analysis of Bovine Bta-miR-494[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 215-223. doi: 10.7668/hbnxb.20194709.

http://www.hbnxb.net/CN/Y2024/V39/I3/215

图/表 9

表1 牛Bta-miR-494及管家基因引物信息

Tab.1 Bovine Bta-miR-494 and housekeeping gene primer information

基因 Gene	引物序列(5'—3') Primer sequence(5'—3')	引物长度/bp Primer length
Bta-miR-494	RT primer:GTCGTATCCAGTGCAGGGTCCG	50
	AGGTATTCGCACTGGATACGACGAGGTT
	F:CCACCATGTGAAACATACACGGG	23
	R:ATCCAGTGCAGGGTCCGAGG	20
18S RNA	F:GTGGTGTTGAGGAAAGCAGACA	22
	R:TGATCACACGTTCCACCTCATC	22

图1 牛Bta-miR-494基因组位置及物种间保守性分析 A.Bta-miR-494在牛基因组中的位置;B.miR-494在物种间的保守性特征;C.miR-494在物种间的系统发育树。

Fig.1 Bovine Bta-miR-494 genomic location and inter-species conservation analysis A.Location of Bta-miR-494 in the bovine genome;B.Conserved features of miR-494 among species;C.Phylogenetic tree of miR-494 among species.

图2 牛Bta-miR-494上游序列CpG岛及启动子特征分析 A.软件EMBOSS CpGplot分析结果;B.软件MethPrimer分析结果。

Fig.2 Characterization of the CpG island and promoter of the upstream sequence of bovine Bta-miR-494 A.Software EMBOSS CpGplot analysis results;B.Software MethPrimer analysis results.

表2 Promoter-2.0预测启动子结果

Tab.2 Promoter-2.0 predicted promoter results

位点 Position	得分 Score	可能性 Likelihood
700	0.593	中
1 600	0.566	中

表3 BDGP:Neural Network Promoter Prediction预测启动子结果

Tab.3 Promoter prediction results by BDGP:Neural Network Promoter Prediction

开始位点 Start	结束位点 End	得分 Score	启动子序列 Promoter Sequence
390	440	0.84	CTTGGTCAGCTCTAAAGCCGGGACACAGAGGTAGGTTGTCTGCATGGTGT
1 053	1 103	0.94	TCCTCTGCTCTAAAGGCCAGCTACAGAGAGAGGGGAGGGCACCATTATAC

图3 Bta-miR-494靶基因预测及GO和KEGG分析结果 A.Bta-miR-494靶基因的GO注释;B.基于3种数据库的Bta-miR-494靶基因预测结果;C.Bta-miR-494靶基因的KEGG通路分析。

Fig.3 Bta-miR-494 target gene prediction and GO and KEGG analysis results A.GO annotation of Bta-miR-494 target gene;B.Bta-miR-494 target gene prediction results based on three databases;C.KEGG pathway analysis of Bta-miR-494 target gene.

图4 牛Bta-miR-494候选靶基因编码的蛋白质互作网络

Fig.4 Interaction network of proteins encoded by Bta-miR-494 candidate target genes

图5 牛Bta-miR-494核心靶基因分析

Fig.5 Analysis of bovine Bta-miR-494 core target genes

图6 Bta-miR-494在成年牛和初生犊牛主要组织中的相对表达量不同小写字母表示差异显著(P<0.05)。

Fig.6 Relative expression of Bta-miR-494 in major tissues of adult cattle and newborn calves Different lowercase letters indicate significant differences(P<0.05).

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